Field of the Invention
The present invention relates to methods and systems for the isolation of DNA on a microfluidic device and the subsequent analysis of the DNA on the microfluidic device. More specifically, embodiments of the present invention relate to methods and systems for the isolation of DNA from patient samples on a microfluidic device and use of the DNA for performing amplification reactions, such as PCR, and detection, such as thermal melt analysis, on the microfluidic device.
Description of Related Art
The detection of nucleic acids is central to medicine, forensic science, industrial processing, crop and animal breeding, and many other fields. The ability to detect disease conditions (e.g., cancer), infectious organisms (e.g., HIV), genetic lineage, genetic markers, and the like, is ubiquitous technology for disease diagnosis and prognosis, marker assisted selection, correct identification of crime scene features, the ability to propagate industrial organisms and many other techniques. Determination of the integrity of a nucleic acid of interest can be relevant to the pathology of an infection or cancer. One of the most powerful and basic technologies to detect small quantities of nucleic acids is to replicate some or all of a nucleic acid sequence many times, and then analyze the amplification products. PCR is perhaps the most well-known of a number of different amplification techniques.
The basic steps of nucleic acid, such as DNA, isolation are disruption of the cellular structure to create a lysate, separation of the soluble nucleic acid from cell debris and other insoluble material, and purification of the DNA of interest from soluble proteins and other nucleic acids. Historically, organic extraction (e.g., phenol:chloroform) followed by ethanol precipitation was performed to isolate DNA. Disruption of most cells is performed by chaotropic salts, detergents or alkaline denaturation, and the resulting lysate is cleared by centrifugation, filtration or magnetic clearing. The DNA can then be purified from the soluble portion of the lysate. When silica matrices are used, the DNA is eluted in an aqueous buffer such as Tris-EDTA (TE) or nuclease-free water.
DNA isolation systems for genomic, plasmid and PCR product purification are historically based on purification by silica. Regardless of the method used to create a cleared lysate, the DNA of interest can be isolated by virtue of its ability to bind silica in the presence of high concentrations of chaotropic salts (Chen and Thomas, Anal Biochem 101:339-341, 1980; Marko et al., Anal Biochem 121:382-387, 1982; Boom et al., J Clin Microbiol 28:495-503, 1990). These salts are then removed with an alcohol-based wash and the DNA eluted in a low ionic strength solution, such as TE buffer or water. The binding of DNA to silica may be driven by dehydration and hydrogen bond formation, which competes against weak electrostatic repulsion (Melzak et al., J Colloid and Interface Science 181:635-644, 1996). Hence, a high concentration of salt will help drive DNA adsorption onto silica, and a low concentration will release the DNA.
Recently, new methods for DNA purification have been developed which take advantage of the negatively charged backbone of DNA to a positively charged solid substrate (under specific pH conditions), and eluting the DNA using a change in solvent pH (ChargeSwitch® technology, Invitrogen, Corp., Carlsbad, Calif.; see, for example, U.S. Pat. No. 6,914,137 and International Published Application No. 2006/004611). Whatman has an alternate technology (FTA® paper) that utilizes a cellulose based solid substrate impregnated with a lysis material that lyses cells, inactivates proteins, but captures DNA in the cellulose fibers, where it is retained for use in downstream applications (see, for example, U.S. Pat. No. 6,322,983).
The use of nuclear extracts was reported in 1983 (Dignani et al., Nucl Acids Res 11:1475-1489, 1983). There are several commercial kits that allow for the selective lysis of a cellular membrane while allowing for the purification of cellular organelles, including nuclei. Sigma and Pierce are two providers of commercial kits. These kits utilize centrifugation for the collection of nuclei. There are two patents that describe purifying nuclei from cells. U.S. Pat. No. 5,447,864 describes using a DNA mesh to capture intact cell nuclei on a membrane to capture nuclei for various applications. A membrane is extended across the forward end of a pipette tip device and the DNA from lysed nuclei is used to capture the remaining intact nuclei. U.S. Pat. No. 6,992,181 B2 describes the use of a CD device for the purification of DNA or cell nuclei. This method requires moving parts and centrifugal force to isolate DNA and or cell nuclei, using a barrier in the channel to impede flow of DNA and nuclei. Both patents describe the capture of nuclei and/or DNA and subsequent steps of washing are also required for their systems.
Various papers have described the capture of nuclei or white blood cells for downstream use in PCR reactions. If only the white blood cells are isolated, the primary inhibitor of PCR reactions, haemoglobin, is removed, yielding higher efficiencies in PCR, (Cheng et al., Nucl Acids Res 24:380-385, 1996). Another approach (Service, Science 282:399-401, 1998) involves mixing blood with a salt solution that lyses the cells. The lysate is then introduced into a chamber containing a glass wall on which DNA binds by charge interaction, while the rest of the sample is ejected. DNA must then be washed and is eluted to another chamber. Another paper describes a microfluidic platform for cell separation and nucleus collection that uses dielectrophoresis (DEP) forces to separate cells in a continuous flow system. After a specific cell was captured a lysis buffer was added and the nucleus of the cell can then be collected to study nuclear proteins. The ability to perform PCR on a single captured nucleus was demonstrated by Li et al. (Eukaryotic Cell 2:1091-1098, 2003). A nucleus extracted by a micropipette was added directly to a PCR reaction for the detection of a specific gene sequence. This paper demonstrates that nuclei added directly to a PCR reaction can be used to deliver DNA template, and assays for specific gene targets can be conducted using nuclei isolated from cells.
The most significant problems with the current technologies are that they require specific buffers for DNA binding and washing, most of which are not compatible with down stream applications such as PCR, and they have a wide range of efficiencies in the overall quantity of DNA that is purified. This can be a significant problem when samples are to be used in microfluidics. The multiple reagents that are typically required for DNA purification would demand that moving parts, such as valves, be constructed into a microfluidic device for the introduction of multiple reagents in a solid phase extraction. In a microfluidic system, solid phase extraction or the use of multiple reagents is complicated and can lead to system failures. Commercial assays that are sold by Sigma and Pierce for cellular organelle purification do not use a filtration process for nuclei capture. Instead, they use centrifugal force to collect the nuclei.
In addition, microfluidic devices have been designed to do cell sorting of whole blood and separating white blood cells from red blood cells and plasma. However, such devices do not maximize the removal of proteins, lipids, and other cellular components that may inhibit PCR in a microfluidic system.
Although the various methods exist to capture nuclei for use in down stream application or to separate specific cells from a sample population, none of these methods describes a device that is capable of extracting cell nuclei and performing PCR assays on the nuclei using the same device. Thus, there is a need to develop improved systems and methods for DNA purification through nuclei isolation and integrated PCR detection of genetic sequences in microfluidic devices.